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  • 1. Banci, Lucia
    et al.
    Blazevits, Olga
    Cantini, Francesca
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lang, Lisa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Luchinat, Claudio
    Mao, Jiafei
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ravera, Enrico
    Solid-state NMR studies of metal-free SOD1 fibrillar structures2014In: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 19, no 4-5, p. 659-666Article in journal (Refereed)
    Abstract [en]

    Copper-zinc superoxide dismutase 1 (SOD1) is present in the protein aggregates deposited in motor neurons of amyotrophic lateral sclerosis (ALS) patients. ALS is a neurodegenerative disease that can be either sporadic (ca. 90 %) or familial (fALS). The most widely studied forms of fALS are caused by mutations in the sequence of SOD1. Ex mortuo SOD1 aggregates are usually found to be amorphous. In vitro SOD1, in its immature reduced and apo state, forms fibrillar aggregates. Previous literature data have suggested that a monomeric SOD1 construct, lacking loops IV and VII, (apoSOD Delta IV-VII), shares the same fibrillization properties of apoSOD1, both proteins having the common structural feature of the central beta-barrel. In this work, we show that structural information can be obtained at a site-specific level from solid-state NMR. The residues that are sequentially assignable are found to be located at the putative nucleation site for fibrillar species formation in apoSOD, as detected by other experimental techniques.

  • 2. Bergh, Johan
    et al.
    Zetterstrom, Per
    Andersen, Peter M.
    Brannstrom, Thomas
    Graffmo, Karin S.
    Jonsson, P. Andreas
    Lang, Lisa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Marklund, Stefan L.
    Structural and kinetic analysis of protein-aggregate strains in vivo using binary epitope mapping2015In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, no 14, p. 4489-4494Article in journal (Refereed)
    Abstract [en]

    Despite considerable progress in uncovering the molecular details of protein aggregation in vitro, the cause and mechanism of protein-aggregation disease remain poorly understood. One reason is that the amount of pathological aggregates in neural tissue is exceedingly low, precluding examination by conventional approaches. We present here a method for determination of the structure and quantity of aggregates in small tissue samples, circumventing the above problem. The method is based on binary epitope mapping using anti-peptide antibodies. We assessed the usefulness and versatility of the method in mice modeling the neurodegenerative disease amyotrophic lateral sclerosis, which accumulate intracellular aggregates of superoxide dismutase-1. Two strains of aggregates were identified with different structural architectures, molecular properties, and growth kinetics. Both were different from superoxide dismutase-1 aggregates generated in vitro under a variety of conditions. The strains, which seem kinetically under fragmentation control, are associated with different disease progressions, complying with and adding detail to the growing evidence that seeding, infectivity, and strain dependence are unifying principles of neurodegenerative disease.

  • 3. Byström, Roberth
    et al.
    Andersen, Peter M.
    Grobner, Gerhard
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    SOD1 Mutations Targeting Surface Hydrogen Bonds Promote Amyotrophic Lateral Sclerosis without Reducing Apo-state Stability2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 25, p. 19544-19552Article in journal (Refereed)
    Abstract [en]

    In good accord with the protein aggregation hypothesis for neurodegenerative diseases, ALS-associated SOD1 mutations are found to reduce structural stability or net repulsive charge. Moreover there are weak indications that the ALS disease progression rate is correlated with the degree of mutational impact on the apoSOD1 structure. A bottleneck for obtaining more conclusive information about these structure-disease relationships, however, is the large intrinsic variability in patient survival times and insufficient disease statistics for the majority of ALS- provoking mutations. As an alternative test of the structure- disease relationship we focus here on the SOD1 mutations that appear to be outliers in the data set. The results identify several ALS- provoking mutations whose only effect on apoSOD1 is the elimination or introduction of a single charge, i.e. D76V/Y, D101N, and N139D/K. The thermodynamic stability and folding behavior of these mutants are indistinguishable from the wild-type control. Moreover, D101N is an outlier in the plot of stability loss versus patient survival time by having rapid disease progression. Commonto the identified mutations is that they truncate conserved salt-links and/or H-bond networks in the functional loops IV or VII. The results show that the local impact of ALS- associated mutations on the SOD1 molecule can sometimes overrun their global effects on apo-state stability and net repulsive charge, and point at the analysis of property outliers as an efficient strategy for mapping out new ALS- provoking features.

  • 4.
    Danielsson, Jens
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Awad, Wael
    Saraboji, Kadhirvel
    Kurnik, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lang, Lisa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Leinartaité, Lina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Marklund, Stefan L.
    Logan, Derek T.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Global structural motions from the strain of a single hydrogen bond2013In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 10, p. 3829-3834Article in journal (Refereed)
    Abstract [en]

    The origin and biological role of dynamic motions of folded enzymes is not yet fully understood. In this study, we examine the molecular determinants for the dynamic motions within the beta-barrel of superoxide dismutase 1 (SOD1), which previously were implicated in allosteric regulation of protein maturation and also pathological misfolding in the neurodegenerative disease amyotrophic lateral sclerosis. Relaxation-dispersion NMR, hydrogen/deuterium exchange, and crystallographic data show that the dynamic motions are induced by the buried H43 side chain, which connects the backbones of the Cu ligand H120 and T39 by a hydrogen-bond linkage through the hydrophobic core. The functional role of this highly conserved H120-H43-T39 linkage is to strain H120 into the correct geometry for Cu binding. Upon elimination of the strain by mutation H43F, the apo protein relaxes through hydrogen-bond swapping into a more stable structure and the dynamic motions freeze out completely. At the same time, the holo protein becomes energetically penalized because the twisting back of H120 into Cu-bound geometry leads to burial of an unmatched backbone carbonyl group. The question then is whether this coupling between metal binding and global structural motions in the SOD1 molecule is an adverse side effect of evolving viable Cu coordination or plays a key role in allosteric regulation of biological function, or both?

  • 5.
    Danielsson, Jens
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Inomata, Kohsuke
    Murayama, Shuhei
    Tochio, Hidehito
    Lang, Lisa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Shirakawa, Masahiro
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pruning the ALS-Associated Protein SOD1 for in-Cell NMR2013In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 135, no 28, p. 10266-10269Article in journal (Refereed)
    Abstract [en]

    To efficiently deliver isotope-labeled proteins into mammalian cells poses a main challenge for structural and functional analysis by in-cell NMR. In this study we have employed cell-penetrating peptides (CPPs) to deliver the ALS-associated protein superoxide dismutase (SOD1) into HeLa cells. Our results show that, although full-length SOD1 cannot be efficiently internalized, a variant in which the active-site loops IV and VII have been truncated (SOD1(Delta IV Delta VII))) yields high cytosolic delivery. The reason for the enhanced delivery of SOD1(Delta IV Delta VII) seems to be the elimination of negatively charged side chains, which alters the net charge of the CPP-SOD1 complex from neutral to +4. The internalized SOD1(Delta IV Delta VII) protein displays high-resolution in-cell NMR spectra similar to, but not identical to, those of the lysate of the cells. Spectral differences are found mainly in the dynamic beta strands 4, 5, and 7, triggered by partial protonation of the His moieties of the Cu-binding site. Accordingly, SOD1(Delta IV Delta VII) doubles here as an internal pH probe, revealing cytosolic acidification under the experimental treatment. Taken together, these observations show that CPP delivery, albeit inefficient at first trials, can be tuned by protein engineering to allow atomic-resolution NMR studies of specific protein structures that have evaded other in-cell NMR approaches: in this case, the structurally elusive apoSOD1 barrel implicated as precursor for misfolding in ALS.

  • 6.
    Danielsson, Jens
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kurnik, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lang, Lisa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cutting Off Functional Loops from Homodimeric Enzyme Superoxide Dismutase 1 (SOD1) Leaves Monomeric beta-Barrels2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 38, p. 33070-33083Article in journal (Refereed)
    Abstract [en]

    Demetallation of the homodimeric enzyme Cu/Zn-superoxide dismutase (SOD1) is known to unleash pronounced dynamic motions in the long active-site loops that comprise almost a third of the folded structure. The resulting apo species, which shows increased propensity to aggregate, stands out as the prime disease precursor in amyotrophic lateral sclerosis (ALS). Even so, the detailed structural properties of the apoSOD1 framework have remained elusive and controversial. In this study, we examine the structural interplay between the central apoSOD1 barrel and the active-site loops by simply cutting them off; loops IV and VII were substituted with short Gly-Ala-Gly linkers. The results show that loop removal breaks the dimer interface and leads to soluble, monomeric beta-barrels with high structural integrity. NMR-detected nuclear Overhauser effects are found between all of the constituent beta-strands, confirming ordered interactions across the whole barrel. Moreover, the breathing motions of the SOD1 barrel are overall insensitive to loop removal and yield hydrogen/deuterium protection factors typical for cooperatively folded proteins (i.e. the active-site loops act as a bolt-on domain with little dynamic influence on its structural foundation). The sole exceptions are the relatively low protection factors in beta-strand 5 and the turn around Gly-93, a hot spot for ALS-provoking mutations, which decrease even further upon loop removal. Taken together, these data suggest that the cytotoxic function of apoSOD1 does not emerge from its folded ground state but from a high energy intermediate or even from the denatured ensemble.

  • 7.
    Danielsson, Jens
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mu, Xin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lang, Lisa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wang, Huabing
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Binolfi, Andres
    Theillet, Franois-Xavier
    Bekei, Beata
    Logan, Derek T.
    Selenko, Philipp
    Wennerström, Håkan
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Thermodynamics of protein destabilization in live cells2015In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, no 40, p. 12402-12407Article in journal (Refereed)
    Abstract [en]

    Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a beta-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 degrees C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized interaction landscape of the cellular interior.

  • 8.
    Danielsson, Jens
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Comparing protein behaviour in vitro and in vivo, what does the data really tell us?2017In: Current opinion in structural biology, ISSN 0959-440X, E-ISSN 1879-033X, Vol. 42, p. 129-135Article in journal (Refereed)
    Abstract [en]

    The recent advancement in moving 'biophysical' analysis of proteins in vivo has finally brought us to a position where we can start to make quantitative comparisons with existing in-vitro data. A striking observation is that protein behaviour in live cells seems, after all, not that different from in test tubes, not even at the level of complex mechanisms like protein aggregation. The example examined in this review is the ALS associated protein SOD1 that apparently retains its in-vitro properties in vivo. Does this mean that the protocols for studying proteins in vivo are somehow oversimplified, or that the macromolecular properties and interplay despite being intrinsically malleable are evolutionary more 'streamlined' than previously anticipated? Whatever the answer may be the time is now right to put these data to critical biological test.

  • 9. Gebauer, Denis
    et al.
    Jansson, Kjell
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hedin, Niklas
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Indications that Amorphous Calcium Carbonates Occur in Pathological Mineralisation-A Urinary Stone from a Guinea Pig2018In: Minerals, ISSN 2075-163X, E-ISSN 2075-163X, Vol. 8, no 3, article id 84Article in journal (Refereed)
    Abstract [en]

    Calcium carbonate is an abundant biomineral that is of great importance in industrial or geological contexts. In recent years, many studies of the precipitation of CaCO3 have shown that amorphous precursors and intermediates are widespread in the biomineralization processes and can also be exploited in bio-inspired materials chemistry. In this work, the thorough investigation of a urinary stone of a guinea pig suggests that amorphous calcium carbonate (ACC) can play a role in pathological mineralization. Importantly, certain analytical techniques that are often applied in the corresponding analyses are sensitive only to crystalline CaCO3 and can misleadingly exclude the relevance of calcium carbonate during the formation of urinary stones. Our analyses suggest that ACC is the major constituent of the particular stone studied, which possibly precipitated on struvite nuclei. Minor amounts of urea, other stable inorganics, and minor organic inclusions are observed as well.

  • 10. Gianni, Stefano
    et al.
    Brunori, Maurizio
    Jemth, Per
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Zhang, Mingjie
    Distinguishing between smooth and rough free energy barriers in protein folding.2009In: Biochemistry, ISSN 1520-4995, Vol. 48, no 49, p. 11825-30Article in journal (Refereed)
    Abstract [en]

    Analysis of curved chevron plots is a powerful tool in investigating protein folding pathways, as the curvatures can be used to gain information about both early and late folding events. When and if accumulation of low-energy intermediates can be ruled out, two different models have classically been applied to describe curved chevron plots, namely , (i) Hammond effects along smooth barrier profiles and (ii) changes in the rate-limiting step between two discrete transition states. The two models lead to very similar numerical solutions, which are generally indistinguishable. This is not surprising, since the smooth barrier assumption approximates barrier profiles with a more complex topology involving multiple local maxima that are too close, or too broad, to yield clear-cut kinks in the chevron data. In this work, we have reconstructed the transition state shifts as a function of protein stability over a wide stability range for three small globular proteins, to screen for fingerprints more sensitive for different barrier profiles. We show that such an analysis represents a valuable test for the discrimination between the two different scenarios.

  • 11.
    Haglund, Ellinor
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kadhirvel, Saraboji
    Lindberg, Magnus O
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Logan, Derek T
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Trimming down a protein structure to its bare foldons: spatial organization of the cooperative unit.2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 4, p. 2731-2738Article in journal (Refereed)
    Abstract [en]

    Folding of the ribosomal protein S6 is a malleable process controlled by two competing, and partly overlapping, folding nuclei. Together, these nuclei extend over most of the S6 structure, except the edge strand β2, which is consistently missing in the folding transition states; despite being part of the S6 four-stranded sheet, β2 seems not to be part of the cooperative unit of the protein. The question is then whether β2 can be removed from the S6 structure without compromising folding cooperativity or native state integrity. To investigate this, we constructed a truncated variant of S6 lacking β2, reducing the size of the protein from 96 to 76 residues (S6(Δβ2)). The new S6 variant expresses well in Escherichia coli and has a well dispersed heteronuclear single quantum correlation spectrum and a perfectly wild-type-like crystal structure, but with a smaller three-stranded β-sheet. Moreover, S6(Δβ2) displays an archetypical v-shaped chevron plot with decreased slope of the unfolding limb, as expected from a protein with maintained folding cooperativity and reduced size. The results support the notion that foldons, as defined by the structural distribution of the folding nuclei, represent a property-based level of hierarchy in the build-up of larger protein structures and suggest that the role of β2 in S6 is mainly in intermolecular binding, consistent with the position of this strand in the ribosomal assembly.

  • 12.
    Haglund, Ellinor
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindberg, Magnus O.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Changes of protein folding pathways by circular permutation. Overlapping nuclei promote global cooperativity.2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 41, p. 27904-27915Article in journal (Refereed)
    Abstract [en]

    The evolved properties of proteins are not limited to structure and stability but also include their propensity to undergo local conformational changes. The latter, dynamic property is related to structural cooperativity and is controlled by the folding-energy landscape. Here we demonstrate that the structural cooperativity of the ribosomal protein S6 is optimized by geometric overlap of two competing folding nuclei: they both include the central beta-strand 1. In this way, folding of one nucleus catalyzes the formation of the other, contributing to make the folding transition more concerted overall. The experimental evidence is provided by an extended set of circular permutations of S6 that allows quantitative analysis of pathway plasticity at the level of individual side chains. Because similar overlap between competing nuclei also has been discerned in other proteins, we hypothesize that the coupling of several small nuclei into extended "supernuclei" represents a general principle for propagating folding cooperativity across large structural distances.

  • 13.
    Haglund, Ellinor
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öman, Tommy
    Department of chemistry, Umeå university.
    Lind, Jesper
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öhman, Anders
    Department of chemistry, Umeå university.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The HD-exchange motions of ribosomal protein S6 are insensitive to reversal of the protein-folding pathway.2009In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 1091-6490, Vol. 106, no 51, p. 21619-21624Article in journal (Refereed)
    Abstract [en]

    An increasing number of protein structures are found to encompass multiple folding nuclei, allowing their structures to be formed by several competing pathways. A typical example is the ribosomal protein S6, which comprises two folding nuclei (sigma1 and sigma2) defining two competing pathways in the folding energy landscape: sigma1 --> sigma2 and sigma2 --> sigma1. The balance between the two pathways, and thus the order of folding events, is easily controlled by circular permutation. In this study, we make use of this ability to manipulate the folding pathway to demonstrate that the dynamic motions of the S6 structure are independent of how the protein folds. The HD-exchange protection factors remain the same upon complete reversal of the folding order. The phenomenon arises because the HD-exchange motions and the high-energy excitations controlling the folding pathway occur at separated free-energy levels: the Boltzmann distribution of unproductive unfolding attempts samples all unfolding channels in parallel, even those that end up in excessively high barriers. Accordingly, the findings provide a simple rationale for how to interpret native-state dynamics without the need to invoke fluctuations off the normal unfolding reaction coordinate.

  • 14. Hu, Hai-Xi
    et al.
    Jiang, Yong-Liang
    Zhao, Meng-Xi
    Cai, Kun
    Liu, Sanling
    Wen, Bin
    Lv, Pei
    Zhang, Yonghui
    Peng, Junhui
    Zhong, Hui
    Yu, Hong-Mei
    Ren, Yan-Min
    Zhang, Zhiyong
    Tian, Changlin
    Wu, Qingfa
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Zhang, Cheng-Cai
    Chen, Yuxing
    Zhou, Cong-Zhao
    Structural insights into HetR-PatS interaction involved in cyanobacterial pattern formation2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 16470Article in journal (Refereed)
    Abstract [en]

    The one-dimensional pattern of heterocyst in the model cyanobacterium Anabaena sp. PCC 7120 is coordinated by the transcription factor HetR and PatS peptide. Here we report the complex structures of HetR binding to DNA, and its hood domain (HetR(Hood)) binding to a PatS-derived hexapeptide (PatS6) at 2.80 and 2.10 angstrom, respectively. The intertwined HetR dimer possesses a couple of novel HTH motifs, each of which consists of two canonical alpha-helices in the DNA-binding domain and an auxiliary alpha-helix from the flap domain of the neighboring subunit. Two PatS6 peptides bind to the lateral clefts of HetR(Hood), and trigger significant conformational changes of the flap domain, resulting in dissociation of the auxiliary alpha-helix and eventually release of HetR from the DNA major grove. These findings provide the structural insights into a prokaryotic example of Turing model.

  • 15. Hubner, Isaac A.
    et al.
    Lindberg, Magnus
    Haglund, Ellinor
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliverberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Shakhnovich, Eugene I.
    Common motifs and topological effects in the protein folding transition state.2006In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, ISSN 0022-2836, Vol. 359, no 4, p. 1075-1085Article in journal (Refereed)
    Abstract [en]

    Through extensive experiment, simulation, and analysis of protein S6 (IRIS), we find that variations in nucleation and folding pathway between circular permutations are determined principally by the restraints of topology and specific nucleation, and affected by changes in chain entropy. Simulations also relate topological features to experimentally measured stabilities. Despite many sizable changes in Φ values and the structure of the transition state ensemble that result from permutation, we observe a common theme: the critical nucleus in each of the mutants share a subset of residues that can be mapped to the critical nucleus residues of the wild-type. Circular permutations create new N and C termini, which are the location of the largest disruption of the folding nucleus, leading to a decrease in both Φ values and the role in nucleation. Mutant nuclei are built around the wild-type nucleus but are biased towards different parts of the S6 structure depending on the topological and entropic changes induced by the location of the new N and C termini.

  • 16. Hörnberg, Andreas
    et al.
    Logan, Derek T
    Marklund, Stefan L
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The coupling between disulphide status, metallation and dimer interface strength in Cu/Zn superoxide dismutase.2007In: J Mol Biol, ISSN 0022-2836, Vol. 365, no 2, p. 333-42Article in journal (Refereed)
  • 17.
    Johansson, Ann-Sofi
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Vestling, Monika
    Zetterström, Per
    Lang, Lisa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Leinartaitė, Lina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Karlström, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Marklund, Stefan L.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cytotoxicity of superoxide dismutase 1 in cultured cells is linked to Zn2+ chelation2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 4, p. e36104-Article in journal (Refereed)
    Abstract [en]

    Neurodegeneration in protein-misfolding disease is generally assigned to toxic function of small, soluble protein aggregates. Largely, these assignments are based on observations of cultured neural cells where the suspect protein material is titrated directly into the growth medium. In the present study, we use this approach to shed light on the cytotoxic action of the metalloenzyme Cu/Zn superoxide dismutase 1 (SOD1), associated with misfolding and aggregation in amyotrophic lateral sclerosis (ALS). The results show, somewhat unexpectedly, that the toxic species of SOD1 in this type of experimental setting is not an aggregate, as typically observed for proteins implicated in other neuro-degenerative diseases, but the folded and fully soluble apo protein. Moreover, we demonstrate that the toxic action of apoSOD1 relies on the protein's ability to chelate Zn(2+) ions from the growth medium. The decreased cell viability that accompanies this extraction is presumably based on disturbed Zn(2+) homeostasis. Consistently, mutations that cause global unfolding of the apoSOD1 molecule or otherwise reduce its Zn(2+) affinity abolish completely the cytotoxic response. So does the addition of surplus Zn(2+). Taken together, these observations point at a case where the toxic response of cultured cells might not be related to human pathology but stems from the intrinsic limitations of a simplified cell model. There are several ways proteins can kill cultured neural cells but all of these need not to be relevant for neurodegenerative disease.

  • 18.
    Kurnik, Martin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hedberg, Linda
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Folding without charges2012In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 15, p. 5705-5710Article in journal (Refereed)
    Abstract [en]

    Surface charges of proteins have in several cases been found to function as structural gatekeepers, which avoid unwanted interactions by negative design, for example, in the control of protein aggregation and binding. The question is then if side-chain charges, due to their desolvation penalties, play a corresponding role in protein folding by avoiding competing, misfolded traps? To find out, we removed all 32 side-chain charges from the 101-residue protein S6 from Thermus thermophilus. The results show that the charge-depleted S6 variant not only retains its native structure and cooperative folding transition, but folds also faster than the wild-type protein. In addition, charge removal unleashes pronounced aggregation on longer timescales. S6 provides thus an example where the bias toward native contacts of a naturally evolved protein sequence is independent of charges, and point at a fundamental difference in the codes for folding and intermolecular interaction: specificity in folding is governed primarily by hydrophobic packing and hydrogen bonding, whereas solubility and binding relies critically on the interplay of side-chain charges.

  • 19.
    Lang, Lisa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kurnik, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Fibrillation precursor of superoxide dismutase 1 revealed by gradual tuning of the protein-folding equilibrium2012In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 44, p. 17868-17873Article in journal (Refereed)
    Abstract [en]

    Although superoxide dismutase 1 (SOD1) stands out as a relatively soluble protein in vitro, it can be made to fibrillate by mechanical agitation. The mechanism of this fibrillation process is yet poorly understood, but attains considerable interest due to SOD1's involvement in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). In this study, we map out the apoSOD1 fibrillation process from how it competes with the global folding events at increasing concentrations of urea: We determine how the fibrillation lag time (τ(lag)) and maximum growth rate (ν(max)) depend on gradual titration of the folding equilibrium, from the native to the unfolded state. The results show that the agitation-induced fibrillation of apoSOD1 uses globally unfolded precursors and relies on fragmentation-assisted growth. Mutational screening and fibrillation m-values (∂ log τ(lag)/∂[urea] and ∂ log ν(max)/∂[urea]) indicate moreover that the fibrillation pathway proceeds via a diffusely bound transient complex that responds to the global physiochemical properties of the SOD1 sequence. Fibrillation of apoSOD1, as it bifurcates from the denatured ensemble, seems thus mechanistically analogous to that of disordered peptides, save the competing folding transition to the native state. Finally, we examine by comparison with in vivo data to what extent this mode of fibrillation, originating from selective amplification of mechanically brittle aggregates by sample agitation, captures the mechanism of pathological SOD1 aggregation in ALS.

  • 20.
    Lang, Lisa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Zetterström, Per
    Brännström, Thomas
    Marklund, Stefan L.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    SOD1 aggregation in ALS mice shows simplistic test tube behavior2015In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, no 32, p. 9878-9883Article in journal (Refereed)
    Abstract [en]

    A longstanding challenge in studies of neurodegenerative disease has been that the pathologic protein aggregates in live tissue are not amenable to structural and kinetic analysis by conventional methods. The situation is put in focus by the current progress in demarcating protein aggregation in vitro, exposing new mechanistic details that are now calling for quantitative in vivo comparison. In this study, we bridge this gap by presenting a direct comparison of the aggregation kinetics of the ALS-associated protein superoxide dismutase 1 (SOD1) in vitro and in transgenic mice. The results based on tissue sampling by quantitative antibody assays show that the SOD1 fibrillation kinetics in vitro mirror with remarkable accuracy the spinal cord aggregate buildup and disease progression in transgenic mice. This similarity between in vitro and in vivo data suggests that, despite the complexity of live tissue, SOD1 aggregation follows robust and simplistic rules, providing new mechanistic insights into the ALS pathology and organism-level manifestation of protein aggregation phenomena in general.

  • 21.
    Leinartaité, Lina
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    ALS-provoking SOD1 mutations benign to the apo state compromise the protein’s ability to retain metalsManuscript (preprint) (Other academic)
  • 22.
    Leinartaité, Lina
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pamment, Ylva
    Kovachev, Petar
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dynamics of the SOD1 functional loops – disorder and fluctuations of the apo state can both be triggered and prevented by metallationManuscript (preprint) (Other academic)
  • 23.
    Leinartaité, Lina
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Saraboji, Kadhirvel
    Nordlund, Anna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Logan, Derek T.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Folding catalysis by transient coordination of Zn2+ to the Cu ligands of the ALS-associated enzyme Cu/Zn superoxide dismutase 12010In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 132, no 38, p. 13495-504Article in journal (Refereed)
    Abstract [en]

    How coordination of metal ions modulates protein structures is not only important for elucidating biological function but has also emerged as a key determinant in protein turnover and protein-misfolding diseases. In this study, we show that the coordination of Zn(2+) to the ALS-associated enzyme Cu/Zn superoxide dismutase (SOD1) is directly controlled by the protein's folding pathway. Zn(2+) first catalyzes the folding reaction by coordinating transiently to the Cu ligands of SOD1, which are all contained within the folding nucleus. Then, after the global folding transition has commenced, the Zn(2+) ion transfers to the higher affinity Zn site, which structures only very late in the folding process. Here it remains dynamically coordinated with an off rate of ∼10(-5) s(-1). This relatively rapid equilibration of metals in and out of the SOD1 structure provides a simple explanation for how the exceptionally long lifetime, >100 years, of holoSOD1 is still compatible with cellular turnover: if a dissociated Zn(2+) ion is prevented from rebinding to the SOD1 structure then the lifetime of the protein is reduced to a just a few hours.

  • 24.
    Lindberg, Magns O.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Haglund, Ellinor
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hubner, Isaac A.
    Shakhnovich, Eugene I.
    Oliverberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Identification of the minimal protein-folding nucleus through loop-entropy perturbations.2006In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, no 11, p. 4083-4088Article in journal (Refereed)
    Abstract [en]

    To explore the plasticity and structural constraints of the protein-folding nucleus we have constructed through circular permutation four topological variants of the ribosomal protein S6. In effect, these topological variants represent entropy mutants with maintained spatial contacts. The proteins were characterized at two complementary levels of detail: by φ-value analysis estimating the extent of contact formation in the transition-state ensemble and by Hammond analysis measuring the site-specific growth of the folding nucleus. The results show that, although the loop-entropy alterations markedly influence the appearance and structural location of the folding nucleus, it retains a common motif of one helix docking against two strands. This nucleation motif is built around a shared subset of side chains in the center of the hydrophobic core but extends in different directions of the S6 structure following the permutant-specific differences in local loop entropies. The adjustment of the critical folding nucleus to alterations in loop entropies is reflected by a direct correlation between the φ-value change and the accompanying change in local sequence separation.

  • 25.
    Lindberg, Magnus O
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Malleability of protein folding pathways: a simple reason for complex behaviour.2007In: Curr Opin Struct Biol, ISSN 0959-440X, Vol. 17, no 1, p. 21-9Article in journal (Refereed)
  • 26.
    Mu, Xin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Choi, Seongil
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lang, Lisa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mowray, David
    Dokholyan, Nikolay V.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Physicochemical code for quinary protein interactions in Escherichia coli2017In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 23, p. E4556-E4563Article in journal (Refereed)
    Abstract [en]

    How proteins sense and navigate the cellular interior to find their functional partners remains poorly understood. An intriguing aspect of this search is that it relies on diffusive encounters with the crowded cellular background, made up of protein surfaces that are largely nonconserved. The question is then if/how this protein search is amenable to selection and biological control. To shed light on this issue, we examined the motions of three evolutionary divergent proteins in the Escherichia coli cytoplasm by in-cell NMR. The results show that the diffusive in-cell motions, after all, follow simplistic physical-chemical rules: The proteins reveal a common dependence on (i) net charge density, (ii) surface hydrophobicity, and (iii) the electric dipole moment. The bacterial protein is here biased to move relatively freely in the bacterial interior, whereas the human counterparts more easily stick. Even so, the in-cell motions respond predictably to surface mutation, allowing us to tune and intermix the protein's behavior at will. The findings show how evolution can swiftly optimize the diffuse background of protein encounter complexes by just single-point mutations, and provide a rational framework for adjusting the cytoplasmic motions of individual proteins, e.g., for rescuing poor in-cell NMR signals and for optimizing protein therapeutics.

  • 27.
    Nordlund, Anna
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Leinartaité, Lina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Saraboji, Kadhirvel
    Aisenbrey, Christopher
    Gröbner, Gerhard
    Zetterström, Per
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Logan, Derek T.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Functional features cause misfolding of the ALS-provoking enzyme SOD12009In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 24, p. 9667-72Article in journal (Refereed)
    Abstract [en]

    The structural integrity of the ubiquitous enzyme superoxide dismutase (SOD1) relies critically on the correct coordination of Cu and Zn. Loss of these cofactors not only promotes SOD1 aggregation in vitro but also seems to be a key prerequisite for pathogenic misfolding in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We examine here the consequences of Zn(2+) loss by selectively removing the Zn site, which has been implicated as the main modulator of SOD1 stability and disease competence. After Zn-site removal, the remaining Cu ligands can coordinate a nonnative Zn(2+) ion with microM affinity in the denatured state, and then retain this ion throughout the folding reaction. Without the restriction of a metallated Zn site, however, the Cu ligands fail to correctly coordinate the nonnative Zn(2+) ion: Trapping of a water molecule causes H48 to change rotamer and swing outwards. The misligation is sterically incompatible with the native structure. As a consequence, SOD1 unfolds locally and interacts with neighboring molecules in the crystal lattice. The findings point to a critical role for the native Zn site in controlling SOD1 misfolding, and show that even subtle changes of the metal-loading sequence can render the wild-type protein the same structural properties as ALS-provoking mutations. This frustrated character of the SOD1 molecule seems to arise from a compromise between optimization of functional and structural features.

  • 28.
    Nordlund, Anna
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    SOD1-associated ALS: a promising system for elucidating the origin of protein-misfolding disease2008In: HFSP Journal, ISSN 1955-2068, Vol. 2, no 6, p. 354-364Article in journal (Refereed)
    Abstract [en]

    Amyotropic lateral sclerosis (ALS) is a neurodegenerative disease linked to misfolding and aggregation of the homodimeric enzyme superoxide dismutase (SOD1). In contrast to the precursors of other neurodegenerative diseases, SOD1 is a soluble and simple-to-study protein with immunoglobulin-like structure. Also, there are more than 120 ALS-provoking SOD1 mutations at the disposal for detailed elucidation of the disease-triggering factors at molecular level. In this article, we review recent progress in the characterization of the folding and assembly pathway of the SOD1 dimer and how this is affected by ALS-provoking mutations. Despite the diverse nature of these mutations, the results offer so far a surprising simplicity. The ALS-provoking mutations decrease either protein stability or net repulsive charge: the classical hallmarks for a disease mechanism triggered by association of non-native protein. In addition, the mutant data identifies immature SOD1 monomers as the species from which the cytotoxic pathway emerges, and point at compromised folding cooperativity as a key disease determinant. The relative ease by which these data can be obtained makes SOD1 a promising model for elucidating also the origin of other neurodegenerative diseases where the precursor proteins are structurally more elusive.

  • 29.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Waltz, an exciting new move in amyloid prediction2010In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 7, no 3, p. 187-8Article in journal (Refereed)
  • 30. Olofsson, Maria
    et al.
    Hansson, Sebastian
    Hedberg, Linda
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Logan, Derek T
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Folding of S6 structures with divergent amino acid composition: pathway flexibility within partly overlapping foldons.2007In: J Mol Biol, ISSN 0022-2836, Vol. 365, no 1, p. 237-48Article in journal (Refereed)
  • 31. Olofsson, Maria
    et al.
    Kalinin, Stanislav
    Zdunek, Janusz
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johansson, Lennart B-A
    Tryptophan-BODIPY: a versatile donor-acceptor pair for probing generic changes of intraprotein distances.2006In: Phys Chem Chem Phys, ISSN 1463-9076, Vol. 8, no 26, p. 3130-40Article in journal (Other academic)
  • 32.
    Sandelin, Erik
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Anna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Andersen, Peter M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Marklund, Stefan S. L.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Amyotrophic lateral sclerosis-associated copper/zinc superoxide dismutase mutations preferentially reduce the repulsive charge of the proteins2007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 29, p. 21230-21236Article in journal (Refereed)
    Abstract [en]

    We provide bioinformatical evidence that protein charge plays a key role in the disease mechanism of amyotrophic lateral sclerosis (ALS). Analysis of 100 ALS-associated mutations in copper/zinc superoxide dismutase (SOD1) shows that these are site-selective with a preference to decrease the proteins' net repulsive charge. For each SOD1 monomer this charge is normally -6. Because biomolecules as a rule maintain net negative charge to assure solubility in the cellular interior, the result lends support to the hypothesis of protein aggregation as an initiating event in the ALS pathogenesis. The strength of the preferential reduction of repulsive charge is higher in SOD1 associated ALS than in other inherited protein disorders.

  • 33.
    Szpryngiel, Scarlett
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Diffuse binding of Zn2+ to the denatured ensemble of Cu/Zn superoxide dismutase 12015In: FEBS Open Bio, E-ISSN 2211-5463, Vol. 5, p. 56-63Article in journal (Refereed)
    Abstract [en]

    The stability and structural properties of the metalloprotein superoxide dismutase 1 (SOD1) are found to depend critically on metal ions. Native SOD1 monomers coordinate one structural Zn2+ and one redox-active Cu2+/1+ to the active site. To do this, the Zn2+ ions need to interact with the SOD1 protein on the denatured side of the folding barrier, prior to the formation of the folding nucleus. In this study, we have examined at residue level the nature of this early Zn2+ binding by NMR studies on the urea denatured-state of SOD1. Nearly complete backbone chemical shift assignments were obtained in 9 M urea at physiological pH, conditions at which NMR studies are scarce. Our results demonstrate that SOD1 is predominantly unstructured under these conditions. Chemical-shift changes upon Zn2+ titration show that denatured SOD1 retains a significant affinity to Zn2+ ions, even in 9 M urea. However, the Zn2+ interactions are not limited to the native metal-binding ligands in the two binding sites, but are seen for all His residues. Moreover, the native Cu2+/1+ ligand H46 seems not to bind as well as the other His residues, while the nearby non-native H43 does bind, indicating that the binding geometry is relaxed. The result suggests that the Zn2+-binding observed to catalyze folding of SOD1 in physiological buffer is initiated by diffuse, non-specific coordination to the coil, which subsequently funnels by ligand exchange into the native coordination geometry of the folded monomer. Altogether, this diffuse binding is a result with fundamental implications for folding of metalloproteins in general.

  • 34. Teilum, Kaare
    et al.
    Smith, Melanie H
    Schulz, Eike
    Christensen, Lea C
    Solomentsev, Gleb
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Akke, Mikael
    Transient structural distortion of metal-free Cu/Zn superoxide dismutase triggers aberrant oligomerization.2009In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 1091-6490, Vol. 106, no 43, p. 18273-8Article in journal (Refereed)
    Abstract [en]

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease linked to the misfolding of Cu/Zn superoxide dismutase (SOD1). ALS-related defects in SOD1 result in a gain of toxic function that coincides with aberrant oligomerization. The structural events triggering oligomerization have remained enigmatic, however, as is the case in other protein-misfolding diseases. Here, we target the critical conformational change that defines the earliest step toward aggregation. Using nuclear spin relaxation dispersion experiments, we identified a short-lived (0.4 ms) and weakly populated (0.7%) conformation of metal-depleted SOD1 that triggers aberrant oligomerization. This excited state emanates from the folded ground state and is suppressed by metal binding, but is present in both the disulfide-oxidized and disulfide-reduced forms of the protein. Our results pinpoint a perturbed region of the excited-state structure that forms intermolecular contacts in the earliest nonnative dimer/oligomer. The conformational transition that triggers oligomerization is a common feature of WT SOD1 and ALS-associated mutants that have widely different physicochemical properties. But compared with WT SOD1, the mutants have enhanced structural distortions in their excited states, and in some cases slightly higher excited-state populations and lower kinetic barriers, implying increased susceptibility to oligomerization. Our results provide a unified picture that highlights both (i) a common denominator among different SOD1 variants that may explain why diverse mutations cause the same disease, and (ii) a structural basis that may aid in understanding how different mutations affect disease propensity and progression.

  • 35.
    Wang, Huabing
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lang, Lisa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Logan, Derek T.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tricking a Protein To Swap Strands2016In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 138, no 48, p. 15571-15579Article in journal (Refereed)
    Abstract [en]

    Despite continuing interest in partly unfolded proteins as precursors for aggregation and adverse gain-of-function in human disease, there is yet little known about the local transitions of native structures that possibly lead to such intermediate states. To target this problem, we present here a protein-design strategy that allows real-time detection of rupture and swapping of complete secondary-structure elements in globular proteins molecular events that have previously been inaccessible experimental analysis. The approach is applied to the dynamic beta-barrel of SOD1, associated with pathologic aggregation in the neurodegenerative disease ALS. Data show that rupture and re-insertion of individual beta-strands do not take place locally but require the SOD1 barrel to unfold globally. The finding questions the very existence of partly unfolded intermediates in the SOD1 aggregation process and presents new clues to the mechanism by which hydrogen bonding maintains global structural integrity.

  • 36. Wood, Rebecca J.
    et al.
    Ormsby, Angelique R.
    Radwan, Mona
    Cox, Dezerae
    Sharma, Abhishek
    Vöpel, Tobias
    Ebbinghaus, Simon
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Reid, Gavin E.
    Dickson, Alex
    Hatters, Danny M.
    A biosensor-based framework to measure latent proteostasis capacity2018In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 287Article in journal (Refereed)
    Abstract [en]

    The pool of quality control proteins (QC) that maintains protein-folding homeostasis (proteostasis) is dynamic but can become depleted in human disease. A challenge has been in quantitatively defining the depth of the QC pool. With a new biosensor, flow cytometrybased methods and mathematical modeling we measure the QC capacity to act as holdases and suppress biosensor aggregation. The biosensor system comprises a series of barnase kernels with differing folding stability that engage primarily with HSP70 and HSP90 family proteins. Conditions of proteostasis stimulation and stress alter QC holdase activity and aggregation rates. The method reveals the HSP70 chaperone cycle to be rate limited by HSP70 holdase activity under normal conditions, but this is overcome by increasing levels of the BAG1 nucleotide exchange factor to HSPA1A or activation of the heat shock gene cluster by HSF1 overexpression. This scheme opens new paths for biosensors of disease and proteostasis systems.

  • 37. Öhman, Anders
    et al.
    Öman, Tommy
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Solution structures and backbone dynamics of the ribosomal protein S6 and its permutant P54-552010In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 19, no 1, p. 183-189Article in journal (Refereed)
    Abstract [en]

    The ribosomal protein S6 from Thermus thermophilus has served as a model system for the study of protein folding, especially for understanding the effects of circular permutations of secondary structure elements. This study presents the structure of a permutant protein, the 96-residue P54-55, and the structure of its 101-residue parent protein S6wt in solution. The data also characterizes the effects of circular permutation on the backbone dynamics of S6. Consistent with crystallographic data on S6wt, the overall solution structures of both P54-55 and S6wt show a β-sheet of four antiparallel β-strands with two α-helices packed on one side of the sheet. In clear contrast to the crystal data, however, the solution structure of S6wt reveals a disordered loop in the region between β-strands 2 and 3 (Leu43-Phe60) instead of a well-ordered stretch and associated hydrophobic mini-core observed in the crystal structure. Moreover, the data for P54-55 show that the joined wild-type N- and C-terminals form a dynamically robust stretch with a hairpin structure that complies with the in silico design. Taken together, the results explain why the loop region of the S6wt structure is relatively insensitive to mutational perturbations, and why P54-55 is more stable than S6wt: the permutant incision at Lys54-Asp55 is energetically neutral by being located in an already disordered loop whereas the new hairpin between the wild-type N- and C-termini is stabilizing.

1 - 37 of 37
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